Cultured endothelial cells constitutively synthesize significant levels of thrombospondin, an extracellular matrix-associated protein with reported anti-anti-angiogenic properties. However, two murine endothelial cell lines, bEND.3 and Py-4-1, which have been immortalized with polyoma T oncogenes and which generate vascular malformations in vivo, produce little or no thrombospondin though bEND.3 (but not Py-4-1) growth is inhibited by the addition of exogenous thrombospondin. In addition, Py-4-1 cells are not growth-inhibited by transforming growth factor-beta, a potent endothelial inhibitor. These results indicate that these two cell lines may be useful tools in understanding the role and mechanism of action of thrombospondin and transforming growth factor-beta in endothelial cell biology. A role for thrombospondin in vascular development is further suggested by the observation of significant differences in the levels of thrombospondin mRNA and protein between capillary and aortic endothelial cells. Transforming growth factor-beta-1 treatment of normal endothelial cells increases steady-state levels of thrombospondin mRNA and protein and results in extensive deposition of thrombospondin into the extracellular matrix. In contrast, transforming growth factor-beta-1 has little effect on thrombospondin levels in the tumorigenic endothelial cell lines. In view of our earlier finding that contact between endothelial cells and mural cells generates activated transforming growth factor-beta-1, and the fact that thrombospondin is present in a fibrillar network around vascular structures in vitro, we speculate that modulation of thrombospondin production and distribution by transforming growth factor-beta may be a physiological process to enjoin stabilization of vessels and cessation of vessel growth.